PROJECT SUMMARY Recent technological advances in transmission electron microscopy of frozen-hydrated specimens (cryo-EM) have made it possible to retrieve the three-dimensional structure of biological macromolecules with near- atomic resolution. In conventional cryo-EM, the thin, transparent protein assemblies are made visible by defocusing the imaging system. The drawback of this method is that the low spatial frequency components of the image, which are essential for identification and classification of the particles, remain heavily attenuated. Poor contrast at low spatial frequencies makes it difficult to reconstruct protein complexes with a molecular weight below 100-200 kDa, or even larger assemblies that exhibit significant structural variability. On the other hand, cryo-EM reconstruction of particles as small as 40 kDa is theoretically possible with an in-focus phase contrast device, such as a Zernike phase plate. Recently, the capabilities of Zernike phase contrast in cryo-EM single particle analysis have been demonstrated with a ?Volta? phase plate, based on a thin amorphous carbon foil. However, a potential for improvement still exists, in particular in achieving a constant, stable phase shift. In this project, we set out to build a Zernike phase plate for transmission electron microscopy (TEM) based on a concept borrowed from the field of atomic physics: coherently controlling the motion of quantum particles with lasers. In this approach, a laser beam focused in the back focal plane of a TEM objective lens creates an effective potential, which retards the phase of the transmitted wave relative to the scattered wave and thus acts as a Zernike phase plate. Since no material objects are inserted in the beam path, a laser-based electron phase plate is not susceptible to electron beam damage. Importantly, it allows for a stable, controllable phase The necessary high-intensity, continuous laser focus will be created by amplifying a laser beam in a near-concentric Fabry?Prot optical resonator with a small mode waist, which we have developed in our recent work. While we have already demonstrated a sustained optical intensity sufficient to retard a 300 keV electron beam by 9, a full 90 phase shift, optimal for a Zernike phase plate, is well within reach with state of the art cavity mirrors. Our numerical simulations show that with a cavity-based laser phase plate, phase contrast extends to sufficiently low spatial frequencies to allow for nearly full-contrast imaging of protein complexes smaller than 5-10 nm. The initial development and characterization of the laser phase plate's efficacy as a tool for protein reconstruction will take place in a custom-built TEM, designed specifically for phase plate development. We will then proceed to build a laser phase plate compatible with standard cryo-EM equipment, with the goal of making the laser-based Zernike phase contrast technology shift to be applied to the transmitted wave. available to the broad structural biology community.